This application describes a research program designed to study the membrane components and mechanism of methotrexate (MTX) transport (via the 5-methyltetrahydrofolate system) in L1210 cells. The results will contribute to an understanding of how MTX enters cells during chemotherapy. The MIX-binding activity of intact L1210 cells will be quantitated and characterized with regard to substrate specificity, pH-and ion-dependence, and response to sulfhydryl inhibitors and modulators of cyclic AMP levels. Photoaffinity and chemical labeling agents will also be employed to probe the binding site of the carrier protein, and to identify the protein during its purification. Studies on the mechanism of MTX transport will provide further evidence for the obligatory exchange of extracellular MTX for intracellular anions.